DESCRIPTION: (Investigator's abstract) Our long-term goal is to understand the pathophysiology of thrombosis to improve its diagnosis and treatment. This project extends research on the intrinsic coagulation pathway and its regulation by anti-thrombotic plasma Protein S. Based on our recent discoveries, we hypothesize that certain HDL subspecies as well as the plasma glycolipid, glucosyl ceramide (GlcCer), enhance expression of activated protein C (APC) and protein S anticoagulant activity by enhancing specific cleavages in factor Va. To identify and characterize plasma HDL subspecies with greater anticoagulant cofactor activity, HDL subfractions will be purified and characterized (Aim 1). To characterize molecular mechanisms by which GlcCer enhances protein S-dependent anticoagulant activity, various coagulation assays will define the procoagulant and/or anticoagulant activities of GlcCer (Aim 2). For protein S anticoagulant activity that is independent of APC, we hypothesize that residues 47-72 and 620-635 of protein S directly bind factor Xa and factor Va, respectively, thereby contributing to "protein S direct" activity. To characterize protein S binding to factor Va and/or factor Xa, we will use recombinant full-length and truncated protein S mutants for functional and binding studies of protein-protein and protein-lipid interactions (Aim 3). We hypothesize that subjects with deficiency of "protein S direct" anticoagulant activity are at increased risk for venous and/or arterial thrombosis. To assess "protein S direct" anticoagulant activity in plasma, new clotting assays for protein S direct anticoagulant activity in plasma will be developed and used in case-control studies of 100 venous thrombosis and 250 acute myocardial infarction patients (Aim 4). To characterize factor Va structure-function relationships for factor Xa binding and for proteolytic inactivation, recombinant site-directed mutants of factor Va involving hypothesized factor Xa binding sites (residues 500-506 and 312-323), near the APC cleavage sites at Arg506 and Arg306 in factor Va, will be prepared and factor Va activity and factor Xa binding will be defined (Aim 5). To determine whether factor Va proteolytic inactivation by APC/protein S is caused primarily by (i) loss of factor Xa binding sites or (ii) dissociation of domain A2, we will create novel disulfide bonds that link the A2 domain to the A1 or A3 domain in factor Va mutants based on our factor Va 3-dimensional model. After APC's cleavage at Arg306 in factor Va mutants containing a non-dissociable A2 domain, we will quantitate factor Va activity and factor Xa binding to define the importance of A2 dissociation. The proposed studies of HDL, GlcCer, protein S and factor Va will increase our insights into the regulation of blood coagulation and thrombosis.